| Objective:Cancer is one of the major diseases to human health. Chemotherapy is the main method for cancer therapy. However, failure of chemotherapy mainly results from the effect of multidrug resistance (MDR). In recent years, screening the low toxicity and efficient reversal agents of P-gp is the hotspot.Chinese herb has low cost, small side effects and also have millennium theory. The treatment of human body is often multi-channel and multi-target. So it becomes a new research direction of multidrug resistance in recent years. In this study, we selected human colon carcinoma cell line(Caco-2) and human gastic carcinoma cell line resistant to cisplatin(SGC-7901/DDP)to screening the different chemical extacts of Herba Rabdosiae Rubescentis, and explore the reverse effect of Herba Rabdosiae Rubescentis's different extracts. This sthdy is aims to find a new type of MDR reversal agents, has low toxicity, and screening the active ingredients from Herba Rabdosiae Rubescentis provide a theoretical basis.Methods:(1) By reflux extraction, Soxhlet extraction and decocting method to extract Herba Rabdosiae Rubescentis's different chemical extracts. Using different polar solvents (petroleum ether, chloroform, ethyl acetate, butanol, water) as the polarity order extract from low to high, vacuum distillation to remove the solvent, and obtain Herba Rabdosiae Rubescentis's different chemical extracts.(2) Selected Caco-2 as screening model, and use fluorescence microplate reader to detect the accumulation of Caco-2's Rho-123, its intervented by Herba Rabdosiae Rubescentis's different chemical extracts. Identified the effctive Herba Rabdosiae Rubescentis's different chemical extracts inhibitors of P-gp initially; MTT assay was used to detect the cytotoxicity of Herba Rabdosiae Rubescentis's different chemical extracts in Caco-2 cells, and detect the cytotoxicity of ADM combined with or without Herba Rabdosiae Rubescentis's different chemical extracts in Caco-2 cells, and calculated Q values. Using flow cytometry detect the accumulation of Rho-123 and ADM in Caco-2 cells, and initially set the active chemical extracts of Herba Rabdosiae Rubescentis to revrese the P-gp;(3) Using HPLC measured the active chemical extracts of Herba Rabdosiae Rubescentis's content of oridonin;(4) The active chemical extracts of Herba Rabdosiae Rubescentis were screened by Caco-2 cells are studied on SGC-7901 and SGC7901/DDP cells, and further validated the active chemical extracts's reversal activity. Results:(1) By reflux extraction, Soxhlet extraction and decoction continuous cooking method extracted Herba Rabdosiae Rubescentis's petroleum ether, chloroform, ethyl acetate, butanol and water extracts, the rate of extraction was 0.96%,2.46%,0.36%,1.62% and 4.84%;(2) Rhodamine accumulation experiments showed chloroform, ethyl acetate, butanol, water extracts of Herba Rabdosiae Rubescentis treated Caco-2 cells with 1 h and 72h, the increation of fluorescence intensity are significant difference between the control group. However, the extract of petroleum ether is not difference between the control group;(3) In the context of non-cytotoxic, chloroform, ethyl acetate, water extracts of Herba Rabdosiae Rubescentis are increased the accumulation of Rho-123 and ADM in Caco-2 cells. But, the extract of butanol can not increase the fluorescence intensity of Rho-123 and ADM. The ethyl acetate extract and chloroform extract of Herba Rabdosiae Rubescentis combined with 1μg/mL ADM treated Caco-2 cells, the combined effect of two drugs is synergy, the extract of water position combined with 1μg/mL ADM treated Caco-2 cells, the combined effect of two drugs is addition. But, the extract of butanol, its combined effect is antagonism. So, we decided the extracts of chloroform, ethyl acetate, and water as the object of study at follow-up experiments;(4) Using HPLC measured the different chemical extracts of Herba Rabdosiae Rubescentis's content of oridonin. We measured the chloroform, ethyl acetate and butanol extracts of Herba Rabdosiae Rubescentis's the contents of oridonin were 0.5%,1.05% and 0.58%, the extracts of water does not contain oridonin;(5) The MTT assay indicated that the IC50 of SGC-7901 and SGC-7901/DDP were treated with DDP are 6.53and 18.11, the ration of resistance is 2.77; The IC50 of SGC-7901 and SGC-7901/DDP were treated with ADM are 0.914 and 3.78, the ration of resistance is 4.14. Using verapamil, oridonin (2.4μM,4.8μM), extract of chloroform (20μg/mL,100μg/mL), extract of ethyl acetate (20μg/mL, 100μg/mL) and extract of water are treated SGC7901/DDP cells, the IC50 are:3.20, 3.73,3,49,7.22,6.46,7.91,5.75,12.28,11.06, and the ration of reversion are 5.65,4.86,5.18,2.51, 2.80,2.29,3.15,1.47,1.64;(6) In the context of non-cytotoxic, oridonin, the extracts of chloroform, ethyl acetate, and water are increase the accumulation of Rho-123 and ADM in SGC-7901/DDP, and the result of ethyl acetate's and chloroform's extracts are better than oridonin. Oridonin, the ethyl acetate's and chloroform's extracts of Herba Rabdosiae Rubescentis combined with 1μg/mL ADM treated SGC-7901/DDP cells, the combined effect of two drugs is synergy, and the result of ethyl acetate's extracts is better than oridonin and the chloroform's extracts.Conclusion:Non-toxic doses of chloroform's, ethyl acetate's, and water's extracts could significantly inhibit the role of P-gp, and increase the sensitivity of Caco-2 and SGC-7901/DDP cells to chemotherapeutic drugs. It also can reverse the multidrug resistance phenomenon. The effective componnents of Herba Rabdosiae Rubescentis could reverse multidrug resistance is not only oridonin, there are other components. |
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